Systemic Lupus Erythematosus (SLE) is a complex, polygenic disease. The delayed onset and variable pentetrance of SLE support the hypothesis that disease pathogenesis is a multi-step process. The highly variable clinical presentation of human SLE makes the study of this process difficult in patients. Here, we take advantage of a tractable and simplified genetic system, the CD45E613R mouse, to interrogate SLE pathogenesis. These mice express a single amino acid substitution that results in constitutive CD45 phosphatase activity. Mirroring the variable presentation of human SLE, the CD45E613R phenotype is extremely sensitive to genetic background. On a hybrid 129-C57Bl/6 (B6) background, CD45E613R mice develop anti-dsDNA antibodies and immune complex-mediated glomerulonephritis (GN). However, on a B6 genetic background mice lack autoantibodies despite biochemical hyper-responsiveness to receptor stimulation while BALB/c CD45E613R mice develop high-titer anti-dsDNA but no GN. We have taken advantage of the genetic separation of cellular hyper-responsiveness, autoantibody production, and end-organ damage to search for genetic modifiers required for disease. Here, we focus on the mechanisms governing the first two steps in SLE pathogenesis: Loss of tolerance to self and peripheral amplification of this autoreactivity. The goal of Aim 1 is to define the contributions of two novel loci we recently identified, Wam1 and Wam2, to autoantibody production. Congenic mice will be generated to test how these loci regulate the threshold for tolerance in the context of hyperactive CD45 phosphatase activity. In Aim 2, we evaluate the contributions of a promising candidate gene in Wam1, Toll Like Receptor (TLR) 9, to disease pathogenesis. Molecular modeling of the TLR9 polymorphisms indicate they map to leucine rich repeats in the DNA ligand binding domain. Our preliminary data indicate differential responses to TLR9 stimulation between B6 and BALB/c mice. These differences are further modulated by the CD45E613R mutation, indicating a novel interaction between CD45 and TLR9 signaling networks. We will use genetic and mixed bone-marrow chimera approaches to define the impact of TLR9 on autoantibody production and identify the cellular compartments in which it must function in CD45E613R mice. The focus of Aim 3 is to interrogate the cellular and molecular mechanisms by which the TLR9 polymorphisms and the CD45E613R mutation regulate tolerance. We will evaluate whether the observed differences are due to altered interaction with ligand, perturbations in intracellular trafficking, and/or altered signal transduction networks. Results of these studies will not only help elucidate the mechanisms governing tolerance, but should provide new insights into how the adaptive and innate arms of the immune system interact in systemic autoimmunity. This is a topic of significant importance in the pathogenesis of autoimmune disease in humans and has broad impact given the growing interest in targeting TLRs in the treatment of autoimmune disease and as an adjuvant in vaccine strategies.